Received December 23, 2017; Revised March 6, 2018; Accepted March 13, 2018.

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Abstract

The sugar beet cyst nematode, Heterodera schachtii is a well known pathogen on Chinese cabbage in the highland fields of Korea. However, a race of cyst forming nematode with close morphological resemblance to H. trifolii was recently isolated from the same Chinese cabbage fields. Morphological species differentiation between the two cyst nematodes is challenging, with only minor differences between them. Thus, this study described the newly intercepted H. trifolii population, and reviewed morphological and molecular characteristics conceivably essential in differentiating the two nematode species. A comparison of morphometric measurements of both infective juveniles and vulval cones of cysts showed significant differences between the two cyst nematodes. When total RNA and genomic DNA were extracted from a mixed field population, COI genes and ITS regions were clearly amplified with primers of the two Heterodera species, suggesting that Heterodera population collected from the Chinese cabbage field consisted of a mixture of two species. COI and ITS of H. trifolii were predominantly amplified from nucleotides prepared from H. trifolii monoxenic population whereas those of H. schachtii were strongly detected in H. schachtii monoxenic cultures. Thus, this study confirms the coexistence of the two species in some Chinese cabbage fields; and the presence of H. trifolii in Korea is reported here for the first time.

H. schachtii and H. trifolii have similarities in host range and thus, coexistence as mixed populations on the same host is possible (Steele and Whitehand, 1984). Distinguishing the two species from each other especially at field level may not be feasible since both exhibit similar morphological body characteristics and symptoms of damage. However, there exist some minor morphological and morphometrical differences useful in distinguishing all species of the H. schachtii sensu stricto group from each other at laboratory level (Amiri et al., 2002).

The SBCN has been identified as one of the serious pests of economic importance on Chinese cabbage production in Korea (Lee et al., 2013). Reports indicate increasing dissemination and infestation rates within the highland Chinese cabbage growing areas of Gangwon-do province (from 11.2 ha in 2011 to 70 ha in 2015) (Kwon et al., 2016). However, a race of cyst nematode with close morphological resemblance to H. trifolii was recently isolated from the same Chinese cabbage fields manifesting symptoms of damage similar to H. schachtii. Chinese cabbage, Brassica rapa chinensis L. (Bae-chu) is a highly valued economic crop and the leading vegetable in both production and consumption in Korea (Chang et al., 2008; Kim et al., 2014). Thus, pest and disease management is very crucial, and efforts to combat cyst nematodes are underway (Kim et al., 2016). However, correct species identification of plant parasitic nematodes is a vital milestone preceding successful nematode control and management strategies. Therefore, this study aimed at providing a detailed morphological, morphometric and molecular description of the newly intercepted H. trifolii population in Korea and review characteristics conceivably essential in distinguishing the two nematode species.

Materials and Methods

Cyst nematode populations

Samples of H. trifolii (YS503) and H. schachtii (GC147) populations were taken from Jeongseon cabbage fields (Table 1), as part of a survey to determine the dispersal rate of cyst nematodes in the infested cabbage fields based on field symptoms of stunted growth, yellowing and wilting of Chinese cabbage plants. YS503 and GC147 were subsequently cultured and maintained as monoxenic cultures on Chinese cabbage in pots at National Academy of Agricultural Science laboratories, Wanju, Korea. Mixed populations (TB372) were sampled from Chinese cabbage fields of Taebaek. Cysts were extracted from infested field soil samples and Chinese cabbage roots using modified Cobb’s decanting and sieving method (EPPO, 2013). Cysts were recovered from a 250 μm sieve and filtered through Whatman® 150 mm filter paper (GE Healthcare Life Sciences, Marlborough, USA) before handpicking using fine forceps under a stereomicroscope Nikon SMZ 1000 (Nikon, Tokyo, Japan). Infective second stage juveniles (J2s) were extracted from the soil using a modified Baermann funnel method (EPPO, 2013).

Morphological identification

Twenty J2 specimens of each species emerging from monoxenic cultured cysts (H. trifolii YS503 and H. schachtii GC147 populations) were heat-killed on temporary slides and directly observed or first fixed and mounted in pure glycerin (Seinhorst, 1959). Ten vulval cones were prepared from undamaged cysts using a method described in Van Bezooijen (2006). Undamaged cysts were soaked in water before vulval cones were excised and transferred into a drop of glycerin-gelatin mixture. Measurements and pictures were made using Zeiss imager Z2 microscope (Carl Zeiss, Gottingen, Germany), fitted with a software AxioVision; Material Science Software for Research and Engineering (Carl Zeiss, Gottingen, Germany), and identification was made based on the key provided by Wouts and Baldwin (1998). All measurements were recorded in μm. Standard deviation and measurement range were calculated and recorded for each arithmetic mean.

Molecular characterization

For molecular characterization, two procedures were performed using species specific and universal primers, respectively. Sequences of COI, ITS and beta-tubulin of the two Heterodera species were compared and species-specific primers were designed for COI and ITS, while conserved regions of beta-tubulin were selected for positive control amplification (Supplementary Table 1, Supplementary Fig. 1). Total RNA and genomic DNA were extracted from cysts with TRI Reagent® (MRC, Cincinnati, OH, USA) according to the manufacturer’s protocol with some modifications. Six cysts (from TB372 population), or individual cyst (from YS503 and GC147 populations) were transferred into an eppendorf tube and completely homogenized with 200 μl TRI Reagent® (MRC) using a bullet blender (Bertin Technologies, Montigny-le-Bretonneux, France). After centrifugation at 12,000 × g for 15 min at 4oC, the aqueous phase and interphase containing total RNA and genomic DNA, respectively, were obtained. After nucleic acid extraction, DNase I was not treated for genomic DNA conservation. First strand cDNA was synthesized from 250 ng total RNA using Superscript™ III reverse transcriptase (Invitrogen, Carlsbad, CA, USA), by priming with oligo (dT). Thirty five cycles of PCR amplifications were performed using Ex Taq DNA polymerase (Takara Korea Biomedical Inc., Seoul, Korea), at 98°C for 10 s, 55°C for 30 s, and at 72°C for 30 s. PCR products were visualized in the 1.5% agarose gel.

For COI and ITS sequencing, DNA was extracted from cysts following a method described by Iwahori et al (2000). Briefly, 10 cysts from each monoxenic population were separately transferred into a drop of water on a glass slide and crushed gently with a piece of filter paper (smaller than 2 mm × 2 mm in size) and small tweezers, by exertion of pressure onto the filter paper. The crushed cyst contents were then transferred into the 0.2 ml PCR tubes containing Proteinase K solution (2 M KCl2, 10 mM Tris HCl, 1 M MgCl2, 10% [w/v] Triton-X 100, 20.6 mg/ml Proteinase K) and DNA was extracted using PCR cycler (PTC-200, MJ Research, CA, USA), at 60°C for 30 minutes and at 94°C for 10 minutes of the lysis process. Polymerase chain reaction (PCR) was then performed to amplify ITS rRNA (Joyce et al., 1994) and COI gene (Derycke et al., 2005) of the 2 cyst-forming nematodes, H. trifolii and H. schachtii, using the universal primer sets as indicated in Supplementary Table 2. The PCR reaction mixture composed of 15 μl PCR premix (Ready-2×-Go with Taq plus; Nanohelix™, Daejeon, Korea), 4 μl template DNA, 0.5 μl forward primer, 0.5 μl reverse primer, and 30 μl sterilized deionized water. PCR reaction was conducted with a thermal cycler (PTC-200, MJ Research) and the program was as follows: 35 cycles of 1 min at 94°C, 1 min at 56°C, and 2 min at 72°C for amplification of ITS rRNA, and 35 cycles of 30s at 94°C, 30s at 53°C, and 45s at 72°C for COI gene. The PCR products were confirmed with electrophoresis equipments (Mupid-eXu, Advance, Tokyo, Japan) and gel documentation system (Major Science, New Taipei City, Taiwan).

Phylogenetic analysis

The PCR products were purified using PCR purification kit (PCR and Gel clean up, Macherey-Nagel, Duren, Germany) with a provided manual. DNA sequence analysis (n=10) of two population was done at GenoTech Corporation (Dajeon, Korea). Outgroup taxa for each dataset was taken based on references from previous studies (Sekimoto et al., 2017; Subbotin et al., 2001; Vovlas et al., 2015). The newly obtained sequences of ITS rRNA and COI gene were aligned using ClustalX 1.83 program with datasets of ‘Schachtii’ group species, registered in National center for biotechnology information. Bayesian analysis of the sequence datasets was performed using MrBayes 3.1.2 (Kang et al., 2016; Sekimoto et al., 2017; Vovlas et al., 2015). The general time reversible substitution model (GTR+I+G) was chosen for this analysis and was run with four chains for 1 × 106 generations. The posterior probability of phylogenetic tree was estimated with MCMC (Markov Chain Monte Carlo) method and consensus tree was generated with a 50% majority rule. The trees were edited and visualized using Dendroscope 3.5.7. Intraspecific and interspecific sequence variation was analyzed using Mega 6.0 program (Tamura et al., 2013).

Statistical analysis

Morphometric data of both J2s and vulval cones of the two nematode species were subjected to analysis of variance according to the general linear model procedure using the SAS statistical package (SAS Institute Inc. Cary, North Carolina, USA). Arithmetic means were compared using Tukey’s HSD (Honest Significant Difference) test at P ≤ 0.05 to test the differences or similarities between measurements of the different morphological characters used in identification. Additionally, arithmetic means, standard deviation and measurement range were calculated for all morphometric characters and comparisons with morphometrics extracted from other similar studies were made (Supplementary Table 2, 3, 4 and 5).

Morphometrical comparisons between H. trifolii and H. schachtii

Statistical comparisons of the morphometric data of J2s and cyst cones between the two species (H. trifolii and H. schachtii) are listed in Table 2 and 3, respectively. The mean body length of H. trifolii J2s is significantly larger than H. schachtii. Correspondingly, lip width, maximum body width, anal body width, stylet length, tail length, hyaline length and distances from anterior end to excretory pore, anterior end to the median bulb valve, anterior end to the pharyngeal-intestine junction and anterior end to the end of pharyngeal gland overlap are significantly larger in H. trifolii compared to H. schachtii. However, lip height and the ratios: body length to the distance from anterior end to median bulb valve and tail length to hyaline length do not significantly differ between the 2 species. Contrary to J2 body length, cyst length and width do not differ significantly between H. trifolii and H. schachtii. However, fenestra length and width, vulval slit length, vulval bridge width and underbridge width show significant differences between H. trifolii and H. schachtii. A comparison with morphometric measurement ranges extracted from other similar studies is shown in Supplementary Table 2, 3, 4 and 5 for H. trifolii and H. schachtii, respectively.

Molecular characterization

The two procedures involving the use of two different primer sets yielded positive results. When nucleic acids were extracted from a group containing mixed field population (TB372), both COI genes and ITS regions were clearly amplified with primers of the two Heterodera species (Fig. 3, see lanes of group samples), suggesting that Heterodera population collected from the Chinese cabbage field does not consist of a single species, but a mixture of two species, H. trifolii and H. schachtii. To identify the species, nucleic acids were prepared from 3 individuals isolated from monoxenic cultures (YS503 and GC147). In individual 1 and 2 (YS503), COI and ITS of H. trifolii were predominantly amplified with H. trifolii-specific primer sets (Fig. 3, see lanes of individual 1 and 2), whereas those of H. schachtii were strongly detected in individual 3 (GC147) after amplification with H. schachtii-specific primer sets (Fig. 3, see lanes of individual 3), suggesting that individual 1 and 2 are H. trifolii, while individual 3 is H. schachtii. These results further confirm the mixture of the two species of Heterodera in the Chinese cabbage fields of Korea.

Phylogeny: ITS rRNA and COI gene

ITS region and COI gene were successfully amplified as indicated in Fig. 4 from YS503 and GC147 population and their sequence information was also obtained and submitted to the GenBank data base under accession numbers; MG682350, MG682348 for H. trifolii and MF043911, KY775592 for H. schachtii. ITS sequence dataset included 22 sequences of ‘Schachtii’ group species of the genus Heterodera, containing newly obtained sequences in Korea. The phylogenetic tree as inferred from Bayesian analysis of the dataset with GTR+I+G substitution model is shown in Fig. 5. The result showed that YS503 (MG682350) population was indistinguishable from others because the resolving power of the clade was very low. Thus, analysis of intraspecific and interspecific sequence variation of ITS rRNA was not conducted. On the other hand, GC147 population belongs to H. schachtii clade according to Fig. 5. Intraspecific sequence variation of ITS rRNA between GC147 (MF043911) population and the published H. schachtii sequence data (EF611118) was 0.0–0.3% while interspecific variations between GC147 (MF043911) population and closely related species, H. glycines (LC208697) and H. medicaginis (AF274391) were 2.0% and 2.1%, respectively. In this analysis, GC147 population was identified as H. schachtii.

The morphometric analysis of the J2s and cyst cones confirms earlier reports that H. trifolii can be differentiated from H. schachtii based on biometric data (Amiri et al., 2003; Mulvey and Golden, 1983; Turner and Subbotin, 2013; Wouts and Baldwin, 1998). In general, the biometric data of H. trifolii J2s is significantly higher than that of H. schachtii (Ambrogioni and Irdani, 2001; Steele and Whitehand, 1984). More notably, total body length, stylet length and Tail length have always been highlighted as the main measurements essential in differentiating between the two species (Steele and Whitehead, 1984); with reports that J2s of H. trifolii are longer (> 500 μm) than H. schachtii J2 (< 500) (Turner and Subbotin, 2013; Wouts and Baldwin, 1998). Our study further points out more detailed significant differences among most of the measured J2 characters between the two species. Thus, a detailed morphometric analysis of the various J2 characters is integral for reliable differentiation of the two nematode species. Contrary, cysts of both species are similar in size. However, Fenestra and vulval slit length are significantly higher in vulval cones of H. trifolii and previous studies highlight their usefulness in differentiating between H. trifolii and H. schachtii in mixed populations (Abawi et al., 1973). A comparison between morphometric data of the described H. trifolii and H. schachtii populations in Korea and populations from other countries (Italy, Sweden, Netherlands, Germany, USA, Turkey, Jordan and Syria) showed remarkable similarities in most of the measurements, though H. trifolii populations from Germany are relatively longer compared to other populations especially from USA. Biometric data, especially the general body length of plant parasitic nematodes are known to vary according to several environmental factors, including geographical localities and differences in the host plants between the isolated populations (Tarte and Mai, 1976). This could explain the comparative morphometric measurement differences among the populations used in comparison, as all were isolated from different geographical locations, environmental conditions and host varieties.

The molecular characterization allows the differentiation of H. trifolii from H. schachtii populations. Morphological differentiation of the cyst nematode species belonging to H. schachtii sensu stricto group is not only difficult but also time consuming and requires specialized taxonomic skills (Amiri et al., 2001). Thus, molecular identification has increasingly become common for efficiency purposes (Amiri et al., 2003), and H. schachtii sensu stricto group primers for the ITS region of ribosomal gene have been developed to differentiate the species within the schachtii group from other nematode species (Amiri et al., 2001; Waeyenberge et al., 2009). However, there is limited interspecific variation in the ITS regions of the H. schachtii group and thus, species-specific identification based on sequences of ITS-rRNA genes within the group is sometimes impossible (Amiri et al., 2002). In this study, unlike COI gene highly species-specific amplification, bands of ITS amplified with primer set being species-specific to H. schachtii were faintly detected in individual 1 and 2 putatively identified as H. trifolii (Fig. 3, lanes of Hsch_ITS in individual 1 and 2). As shown in Fig. 2A, 2B, sequences of ITS from the two species are more similar to each other than those of COI, thereby exhibiting non-specific amplification in individual 1 and 2. However, band intensities of Htrifol_ITS were distinguishably stronger than those of Hsch_ITS, and COI genes of H. trifolii were only amplified with Htrifol_COI in individual 1 and 2 samples, suggesting identification of individual 1 and 2 as H. trifolii. In individual 3, both COI gene and ITS region were amplified with only primer sets which are specific to the sequence of H. schachtii, thus strongly showing that individual 3 is H. schachtii. Sequence information of ITS and COI gene analyzed in this study also confirms the coexistence of two Heterodera species in Chinese cabbage fields in Korea (Fig. 3, 4, 5, 6). Generally, although non-specific bands were slightly visualized by amplification with Hsch_ITS primer set, species-specific primer sets designed in this study can be useful for molecular identification of two Heterodera species. Additionally, in the present study, both coding gene (beta-tubulin and COI) and non-coding region (ITS) were efficiently amplified from nucleic acids including genomic DNA and total RNA extracted with TRI Reagent® (MRC) from individual cyst sample. In fact, genomic DNA is technically difficult to be isolated from a single cyst due to its limited size. However, when extraction of both RNA and DNA from respective aqueous phase and interphase was done after homogenization with TRI Reagent® (MRC) without treatment with DNase I, beta-tubulin, COI gene and ITS region were predominantly amplified as shown in Fig. 3. These results also suggest that the modified TRI Reagent® (MRC) manual can be a proper method for molecular identification of nematode species from a single cyst.

Contrastingly, there exist different levels of intraspecific variation in the ITS-rDNA sequences within ‘Schachtii’ group and this has an effect on cluster analysis (Madani et al., 2007). Indeed, the phylogenetic relationships of the ITS sequences showed two sub-branches of the H. schachtii populations; the sequence of H. schachtii (EF611118) population was clustered together with closely related ‘Schachtii’ group species like H. betae (LC208689), H. daverti (KT163232), H. trifolii (LC208683), including the Korean population of H. trifolii (YS503; MG682350). The second sub-branch consisted of four H. schachtii species (LC208693, AY166438, LC208692, EF611107), populations available in the Genbank ( https://www.ncbi.nlm.nih.gov), together with Korean population of the same species (GC147; MF043911). The populations were not grouped according to their geographical location and these results are in agreement with Madani et al. (2007) and Caswell-Chen et al. (1992), who indicated that the genetic similarity of H. schachtii populations is not correlated with their geographical proximity. Indeed in the study done by Madani et al. (2007), sequences of two ITS clone variants from the same population did not cluster together in Maximum Parsimony trees. Nonetheless, Sekimoto et al. (2017) recently showed that H. trifolii can easily be distinguished from H. schachtii using the COI mtDNA gene and certainly our results are in agreement with the findings. The COI mtDNA gene is able to differentiate between the 2 species without a further application of restriction enzymes and therefore, the gene can be considered as a reliable molecular target, with a precise detection of the two species either individually or in mixed populations. Indeed, the phylogenetic analysis of the COI gene sequences precisely grouped H. schachtii and H. trifolii in separate individual sub-branches with bootstrap of 100%. H. schachtii sub-branch consisted of the H. schachtii species; GC147 (KY775592), LC208708, KC172918, CF100386 and CF101076 while H. trifolii sub-branch consisted of H. trifolii species; YS503 (MG682348), LC208699 and KT163239. However, a combinative comparison between morphological, morphometrical and genetic variations is always the most ideal approach in identification and separation of the closely related species (Amiri et al., 2001).

H. schachtii has already been identified as a serious setback in Chinese cabbage fields in Korea, and efforts in devising proper management and control strategies are underway (Kim et al., 2016; Kwon et al., 2016). However this study highlights the coexistence of H. schachtii and H. trifolii in Chinese cabbage fields and thus, management strategies should be formulated to target both pathogens. Both species have a relatively similar host range hence coexistence is not coincidental but a true possibility as noted by Abawi et al (1973). Conclusively, the presence of H. trifolii in Korea is reported here for the first time.

Acknowledgments

This research was carried out with the support of the “Research Program for Agriculture Science and Technology Development (Project No. PJ012640)”, Rural Development of Administration, Republic of Korea. Thanks to Kyoung Hwan Moon, Faisal Kabir, Mungi Jeong, Jeongun Kim, Hyeon Guk Kim, Yoon Suk Cha, Heebeen Na and Hyeon Jeong Ahn for the necessary laboratory technical support.

Fig. 3. PCR patterns of COI gene and ITS region of Heteroderma trifolii and Heteroderma schachtii amplified with species-specific primers designed in this study (M: 100 bp DNA ladder). Primers of Hetero β-tubul were designed at the conserved region of beta-tubulin from the two Heterodera species. The information of each primer is available in and .

Fig. 5. Phylogenetic relationships between Heterodera species as inferred from Bayesian analysis of internal transcribed spacer (ITS) sequence dataset with general time reversible substitution model (GTR+I+G). The newly obtained sequences were represented in bold. Posterior probability values more than 50% are given for appropriate clade.

Fig. 6. Phylogenetic relationships between Heterodera species as inferred from Bayesian analysis of mitochondrial DNA cytochrome c oxidase subunit I (COI) sequence dataset with general time reversible substitution model (GTR+I+G). The newly obtained sequences were represented in bold. Posterior probability values more than 70% are given for appropriate clade.

Tables

Table 1

The nematode populations used in this study

Population

Date

Host plant

Coordinates

GC147

27th Jul 2016

Chinese cabbage

37.3330700, 128.8464880

YS503

16th Aug 2017

Chinese cabbage

37.4397300, 128.8565550

TB372

9th Oct 2017

Chinese cabbage

37.2558180, 128.9830310

Coordinates for GC147 and YS503 represent individual cabbage fields where samples were taken in Jeongseon, respectively while Coordinates for TB372 represent cabbage field where mixed populations were sampled in Taebaek.

Mulvey, RH, and Golden, AM (1983). An illustrated key to the cyst-forming genera and species of Heteroderidae in the western hemisphere with species morphometrics and distribution. J Nematol. 15, 1-59.

Waeyenberge, L, Viaene, N, Subbotin, SA, and Moens, M (2009). Molecular identification of Heterodera spp., an overview of fifteen years of research. Proceedings of the first workshop of the international cereal cyst nematode initiative, pp. 21-23